The long-term objective of this project is to understand the mechanisms of a wide range of functions performed by the actin cytoskeleton. The proposed research will focus on the basic principles that control cell shape and migration, which play an important role in numerous physiological and pathological processes including embryonic development, wound healing, and metastatic invasion. In addition, control of cell shape and migration represents a bottleneck in regenerative medicine with few effective strategies. While rapid advances have been made on identifying and characterizing the molecular components, the fundamental rules governing the responses of these functions to the physical environment remain largely unknown. The study will combine powerful optical, mechanical, chemical, microengineering, and mathematical approaches to address the responses of adherent cells to substrate rigidity and topography, which are known to have profound effects on cell shape and migration, as well as downstream processes including growth and differentiation. The first aim will characterize cellular responses to rigidity, by determining the size scale of the sensor. The experiments will also test a hypothetical universal mechanism for sensing mechanical signals, and investigate the possible role of rigidity sensing in cancer invasion. The second aim will identify key parameters that determine cellular responses to the geometry of adhesive substrates. In addition, the experiments will test potential mechanisms of geometry sensing, including one based on differential tension at adhesion sites and the other on differential microtubule density and/or dynamics. The third aim will probe the mechanism that allows cells to distinguish 3D environments from 2D surfaces. The experiments will address the topographic requirements and the possible involvement of contractile forces, cortical rigidity, and microtubule positioning. The project will take full advantage of microfabricated substrates, in addition to other innovative approaches from photo- modulation, micromanipulation, to image processing. Cells will be manipulated with pharmacological and gene ablation/silencing techniques, and imaged with high-resolution confocal and total internal reflection fluorescence optics. The fundamental principles revealed by these experiments will impact a broad range of medical issues related directly or indirectly to the regulation of cell shape and migration. PUBLIC HEALTH REVELANCE: There is now extensive evidence that physical and topographical signals, including mechanical forces, rigidity, and geometric patterns, have profound effects on cell shape and migration, which in turn affect a wide range of normal and pathological processes from cancer invasion to stem cell differentiation. However, while cellular responses to chemical interactions have been studied extensively, much less is known about their responses to these non-chemical signals, partially due to the limitations in conventional experimental methods. The proposed experiments will apply highly innovative approaches to overcome the barrier, and to obtain crucial information for understanding and treating many health issues related to cell shape control and cell migration.